飞蝗CYP408B1和CYP409A1基因的原核表达
Prokaryotic expression of the CYP408B1 and CYP409A1 genes in Locusta migratoria
高翠娥** 任晓宇 张学尧 张婷婷 张建珍 马恩波 吴海花***
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DOI：10.7679/j.issn.2095-1353.2015.016
作者单位：山西大学应用生物学研究所，太原 030006
中文关键词：飞蝗，细胞色素P450，Real-time PCR，RT-PCR，原核表达
英文关键词： Locusta migratoria, cytochrome P450, Real-time PCR, RT-PCR, prokaryotic expression
中文摘要：
【目的】 细胞色素P450是分布极其广泛的超家族酶，在昆虫内源及外源化合物代谢中发挥着重要的作用。本文分析了飞蝗Locusta migratoria CYP408B1和CYP409A1基因在不同组织部位的表达差异，并对两种蛋白进行原核表达，为其分子特性和生物学功能的深入研究提供基础资料。【方法】 提取飞蝗5龄若虫不同组织部位的总RNA，体外反转录成cDNA，采用Real-time PCR和RT-PCR技术分析飞蝗CYP408B1和CYP409A1在不同组织部位的表达模式，构建表达载体pCW/CYP408B1、pCW/CYP409A1和pAC/CPR，将pCW/CYP408B1和pCW/CYP409A1分别与pAC/CPR在大肠杆菌Escherichia coli BL21（DE3）中进行共表达。【结果】 通过PCR检测，发现CYP408B1和CYP409A1在飞蝗5龄若虫触角、脑、视叶、咽下神经节、胸神经节和附腺中均有表达，其中CYP408B1在附腺中表达量较高。原核表达结果显示，CYP409A1和CPR（NADPH细胞色素P450还原酶）均可表达，蛋白分子量分别约为58 ku和77 ku，但均为包涵体，而CYP408B1未能成功表达。【结论】 本文揭示了飞蝗CYP408B1和CYP409A1在不同组织部位的表达模式，并对YP409A1和CPR进行了原核表达，研究结果为深入探讨飞蝗细胞色素P450基因对杀虫剂的代谢解毒作用提供了实验依据和基础资料。
英文摘要：
[Objectives] Cytochrome P450s are ubiquitous superfamily enzymes that play important roles in the metabolism of endogenous and exogenous compounds in insects. In order to provide a basis for the further study of the molecular properties and biological functions of the CYP408B1 and CYP409A1 genes in Locusta migratoria, we analyzed their expression patterns in different tissues and performed the prokaryotic expression of these two genes. [Methods] Total RNA of different tissues from fifth-instar nymphs of L. migratoria was used to synthesize cDNA using MLV reverse transcriptase. Real-time PCR and RT-PCR were conducted to analyze the mRNA expression patterns of CYP408B1 and CYP409A1. In addition, the constructed expression plasmids pCW/CYP408B1 and pCW/CYP409A1 were co-expressed with pAC/CPR in E. coli BL21 (DE3), respectively. [Results] CYP408B1 and CYP409A1 were expressed in the antenna, brain, optic lobe, subpharygeal ganglion, thoracic ganglia and accessory gland, while CYP408B1 had higher expression levels in the accessory gland by PCR. The prokaryotic expression results show that CYP409A1 and CPR (NADPH-cytochrome P450 reductase) were successfully expressed in inclusion bodies. Their molecular weights were approximately 58 ku and 77 ku, respectively. The recombinant CYP408B1 was not expressed in the E. coli expression system. [Conclusion] The results reveal the expression patterns of CYP408B1 and CYP409A1 in different tissues of L. migratoria and confirm the expression of the recombinant CYP409A1 and CPR by a prokaryotic expression system. These results provide an experimental basis and basic data for further research on the detoxification of insecticides by cytochrome P450 genes in L. migratoria.